The development of aquaculture has generated a significant shift in global food production away from traditional catch production methods. Driven primarily by population increases, as well as a lack of growth in traditional capture fishery production, aquaculture has expanded rapidly to become a major component in the world-wide food production eco-system. Aquaculture is now seen as playing a key role in many emerging economies, by virtue of its potential to contribute to increased food production while helping reduce pressure on fish resources. As noted by the United Nations Food and Agriculture Organization (UNFAO) in its 2016 Report on the State of the World Fisheries and Aquaculture, aquaculture is the fastest growing area of animal protein production and has significantly outpaced traditional capture fishery production. For example, the UNFAO estimates that aquaculture production now accounts for half of all seafood produced for human consumption.
The increasing global population, growing demand for seafood, and limitations on production from capture fisheries will inevitably lead the continued global expansion of aquaculture with its associated risks of disease emergence and spread. Despite the world's growing reliance on aquaculture as a primary source of food production, especially in many developing economies, traditional aquaculture systems present several technical and biological challenges that limit their overall effectiveness.
One major drawback of aquaculture systems is that the aquatic animals are typically placed in high density production systems. This can result in stress from crowding and sub-optimal water quality conditions that provide for easy transmission of disease. In particular, disease outbreaks in aquaculture systems can result in massive losses among aquatic populations, resulting in large economic losses in commercial aquaculture. Indeed, such disease outbreaks have reportedly cost the aquaculture industry tens of billions dollars in the last 20 years.
In the case of shrimp aquaculture, the problem of disease is especially severe. According to the UNFAO, although global aquaculture shrimp production has increased, major producing countries, particularly in Asia, have experienced a significant decline in output as a result of widespread shrimp disease. There are several reasons for this.
First, unlike vertebrates, shrimp lack many of the key components of adaptive and innate immune response mechanisms preventing many traditional methods of inducing or enhancing natural disease resistance.
Second, most of the major pathogenic viruses cause very low level persistent infections that can occur at moderate to very high prevalence in apparently healthy shrimp populations. The majority of shrimp pathogens are transmitted vertically and disease is the result of a massive viral amplification that follows exposure to various forms of environmental or physiological stress. Stressors can include handling, spawning, poor water quality, or abrupt changes in temperature or salinity. Shrimp viruses can also be transmitted horizontally. Once viral loads are high and disease is manifest, horizontal transmission of infection is accompanied by transmission of disease.
Third, shrimp commonly are infected simultaneously or sequentially with multiple viruses, or even different strains of the same virus. This fact poses significant challenges for diagnosis, detection, and pathogen exclusion in aquaculture systems.
As one example among many, white spot syndrome (WSS) is a viral disease caused by white spot syndrome virus (WSSV). WSSV is a major pathogen in shrimp that causes high mortality and huge economic losses in shrimp aquaculture. The WSS virion is a nonoccluded ellipsoid- or bacilliform-shaped enveloped particle about 275 nm in length and 120 nm in width. Its circular double-stranded DNA consists of 300 kbp covering approximately 185 open reading frames (ORFs). WSSV is currently one of the most significant impediments to the economical sustainability and growth of the global crustacean aquaculture trade. It has caused the loss of billions in USD since its original outbreaks in the early 1990s. To date, WSSV outbreaks have been detected in most major shrimp producing areas including Asia, Central, South and North America, Europe and Africa.
Traditional efforts to prevent and treat shrimp pathogens, such as WSSV, have been met with limited success. For example, attempts to reduce environmental and physiological stressors has been limited due to the economic production needs of aquaculture systems as well as a lack of technical expertise and appropriate aquaculture facilities in many developing countries. Other attempts have been made to create and isolate pathogen-free populations for aquaculture.
However, such efforts are slow and require significant expertise and diagnostic capabilities that are prohibitively expensive. Large-scale applications of antibiotics have been applied to shrimp aquaculture, in particular during the production cycle, both in the larval and growth phases. However, the use of antibiotics in aquaculture has been associated with environmental and human health problems, including bacterial resistance, and persistence of the disease in the aquatic environment. The accumulation of antibiotic residues in the edible tissues of shrimp may also alter human intestinal flora and cause food poisoning or allergy problems. Most importantly, antibiotics are ineffective against viruses. Other methods such as the application of immunostimulants or bacteriophage treatments to target specific pathogens have been tried with limited commercial and practical success.
An effective system for the biocontrol of pathogens in aquaculture and other animal systems may be: 1) pathogen (virus)-specific so as to not kill off-target organisms; 2) robust or catalytic in mode of action; 3) stable and not easily lost throughout development of the target animal; 4) efficient to deliver; 5) simple to manage and low cost; and 6) self-sustainable or regenerating.
To that end, methods of regulating gene expression in pathogens may be an avenue to address the concerns addressed above. Regulating gene expression either by increasing expression or decreasing expression of genes responsible for virulence is considered beneficial for treatment of diseases. This is especially important in those diseases in which master regulatory genes have been identified. While a majority of efforts have been extended toward enhancing gene expression, down-regulating specific gene expression is equally important. A naturally occurring gene-silencing mechanism triggered by double-stranded RNA (dsRNA), designated as small interfering RNA (siRNA), has emerged as a very important tool to suppress or knock down gene expression in many systems. RNA interference is triggered by dsRNA that is cleaved by an RNase-III-like enzyme, Dicer, into 21-25 nucleotide fragments with characteristic 5′ and 3′ termini. These siRNAs act as guides for a multiprotein complex, including a PAZ/PIWI domain containing the protein Argonaute2, that cleaves the target mRNA. These gene-silencing mechanisms are highly specific and potent and can potentially induce inhibition of gene expression throughout an organism.
The last two decades have also seen tremendous progress in gene expression technology, including the continued development of both non-viral and viral vectors. The non-viral approach to gene expression involves the use of plasmid DNAs (pDNAs), which have a number of advantages, including ease of use and preparation, stability and heat resistance, and unlimited size. The plasmids do not replicate in mammalian hosts and do not integrate into host genomes, yet they can persist in host cells and express the cloned gene for a period of weeks to months.
One area that has seen renewed interest in the use of inhibitory RNA molecules is infectious diseases, and in particular, pathogens that affect aquaculture populations. Such strategies for the biocontrol of pathogens may include paratransgenesis and/or the application of paratransgenic principles. Paratransgenesis generally refers to systems whereby symbiotic bacteria, or bacteria capable of colonizing the host for a sufficient amount of time to delivery a therapeutic molecule such as a dsRNA, are genetically modified and reintroduced in the pathogen-bearing host or a pathogen-susceptible population, such as shrimp in aquaculture, where they express effector molecules. However, paratransgenesis has several technical limitations. For example, bacteria to be used in paratransgenesis must generally have three key components: an effector molecule that achieves the desired effect; a mechanism to display or excrete the effector molecule; and bacteria that can survive in the host long enough to produce the expected amount of effector molecules and thereby achieve the desired effect in the host. Therefore, finding such suitable bacteria that fit all of these criteria is very difficult.
Paratransgenesis is generally understood as a technique that attempts to eliminate a pathogen from vector populations through transgenesis of a symbiont of the vector. The goal of this technique is to control vector-borne diseases. The first step is to identify proteins that prevent the vector species from transmitting the pathogen. The genes coding for these proteins are then introduced into the symbiont, so that they can be expressed in the vector. The final step in the strategy is to introduce these transgenic symbionts into vector populations in the wild. Characteristics of a successful n order to perform paratransgenesis may include:                The symbiotic bacteria can be grown in vitro easily.        They can be genetically modified, such as through transformation with a plasmid containing the desired gene.        The engineered symbiont is stable and safe.        The association between vector and symbiont cannot be attenuated.        Field delivery is easily handled.        
A paratransgenic system is a system that can achieve paratransgenesis in a target organism. Identification of suitable commensal bacteria that are non-pathogenic to humans or animals among the many organisms that a host may harbor, particularly in their digestive systems, is paramount for the success of a paratransgenic system. For example, the chosen bacteria should be capable of colonizing a wide variety of shrimp species so that they can be deployed in different species and isolated strains.
Furthermore, a well-designed paratransgenic system must also ensure that the effector molecule does not interfere with any critical host process, such as reproduction and the like. Such technical and physiological challenges make the development of paratransgenic systems extremely difficult. Importantly, these technical issues are such that many paratransgenic systems are neither effective nor appropriate as an effective biocontrol strategy, especially in complex organisms like shrimp. These difficulties may also prevent many paratransgenic systems from being appropriately scaled-up to be effective for environmental deployment. Generally, biocontrol means utilizing disease-suppressive microorganisms to eliminate, control or prevent infection, expression and/or transmission of selected pathogens.
The foregoing problems regarding the biocontrol of pathogens in aquaculture and other animal systems may represent a long-felt need for an effective—and economical—solution to the same. While implementing elements may have been available, actual attempts to meet this need may have been lacking to some degree. This may have been due to a failure of those having ordinary skill in the art to fully appreciate or understand the nature of the problems and challenges involved. As a result of this lack of understanding, attempts to meet these long-felt needs may have failed to effectively solve one or more of the problems or challenges here identified. These attempts may even have led away from the technical directions taken by the present inventive technology and may even result in the achievements of the present inventive technology being considered to some degree an unexpected result of the approach taken by some in the field.
As will be discussed in more detail below, the current inventive technology overcomes the limitations of traditional pathogen control systems, while meeting the objectives of a truly effective vector biocontrol strategy.